Syphilis, caused by the spirochetal bacterium Treponema pallidum, continues to play prominently as a sexually transmitted disease. Syphilis pathogenesis also represents a paradigm of bacterial chronicity and immune evasion, but virtually nothing is known about how T. pallidum carries out these enigmatic processes. More specifically, the T. pallidum outer envelope, comprised of a cytoplasmic and outer membrane, must serve as both the physical and functional interface within the human host. Unfortunately, even though the T. pallidum genome has been sequenced, there remains a scarcity of information on the functions of T. pallidum membrane and membrane-associated proteins that likely contribute to the spirochete's complex parasitic strategy. Among the putative membrane proteins, T. pallidum is postulated to encode about 24-35 lipoproteins. Membrane lipoproteins of other bacteria subserve many important physiological roles and also have importance as virulence factors, modular components of ABC-type transporters, protective immune targets, and proinflammatory agonists that evoke robust innate immune responses. However, the functions of the treponemal lipoproteins remain essentially undefined. In a departure from more traditional approaches to T. pallidum research, the proposed study brings together a group of treponematologists, molecular biologists, protein biochemists, and structural biologists to address this important information gap in a novel way. The Specific Aims of this proposal are: (1) To clone and express in E. coli the lipoprotein genes of T. pallidum, with emphasis on expressing high quantities of each polypeptide as a nonacylated (soluble) fusion protein; (2) To purify to homogeneity each fusion protein and perform biophysical assessments of protein conformation; and (3) To obtain protein crystals suitable for X-ray diffraction and solve the three-dimensional structure for each crystallizable lipoprotein. Extensive preliminary data and progress in other structural biology initiatives support the timeliness and feasibility of this project; many state-of-the-art protein structural characterization techniques will enhance overall success. Finally, structural data will be used to formulate new testable hypotheses regarding potential function(s) of the lipoproteins, new avenues of investigation for T. pallidum membrane biology and syphilis pathogenesis that are sorely needed.